Target Team: Monica L., Tyler T., James F. *Target (protein/gene name): Tyrosine Phosphatase *NCBI Gene # or RefSeq#: Gene #: 985934 *Protein ID (NP or XP #) or Wolbachia#: NP_465325.1 *Organism (including strain): Listeria monocytogenes Etiologic Risk Group (see link below): Risk Group 2 (moderate individual risk, low community risk) */Disease Information (sort of like the Intro to your Mini Research Write up):
Figure 1 - Image of L. monocytogenes bacteria.
L. monocytogenes is a food-borne bacterium that leads to listeriosis; it is the third most common foodborne bacterial pathogen. It can be found in raw vegetables, meat, raw or unpasteurized milk, and dairy products containing such milk. Those at greatest risk include newborn babies (in which listeriosis often results in sepsis and/or meningitis), pregnant mothers, elderly, and those with weakened immune systems, such as HIV patients. Symptoms include nausea, fever, and diarrhea, with a mortality rate of 20-30%. Its fatality rates can surpass those of Salmonella. As a Gram-positive facultative anaerobe, L. monocytogenes is able to survive in both aerobic and anaerobic environments. It expresses the protein beta hemolysin, and is thus able to destroy the host's red blood cells. L. monocytogenes contains D-galactose residues on its surface that interact with D-galactose receptors of the host's intestinal cells, allowing the bacteria to move past the intestinal walls into the host's body. Another protein, internalin, also interacts with intestinal cell proteins to allow entry and invasion. Once it has invaded, the bacterium moves swiftly through the host cell and multiplies, often passing through the blood-brain barrier. Link to TDR Targets page (if present): N/A Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):http://www.ncbi.nlm.nih.gov/gene/?term=985934 Essentiality of this protein:http://iai.asm.org/content/79/6/2489.full Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): N/A Complex of proteins?: N/A Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):http://www.jbc.org/content/279/23/24226.long *EC#: 3.1.3.48 Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.3.48 -- Show screenshot of BRENDA enzyme mechanism schematic
Figure 2 - Brenda Reaction Mechanism for Tyrosine Phosphatase protein (EC # 3.1.3.48) from http://www.brenda-enzymes.org/structure.php?show=reaction&id=11748&type=I&displayType=marvin
--- List cost and quantity of substrate reagents, supplier, and catalog #
All reagents are from Sigma-Aldrich: Imidazole: I202-1G ($11.50) = 1 gramPhosphatase Substrate: S0942 ($35.50) = 50 TabletsSodium Chloride: S9625 ($33.90) = 500 gramsDL-Dithiothreitol: D0632 ($41.70) = 1 gramEthylenediaminetetraacetic acid disodium salt dehydrate: ED2SS ($25.70) = 50 gramsBovine Serum Albumin: A8022 ($66.90) = 10 gramsSodium Hydroxide: S5881 ($51.20) = 500 grams Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: Not available (will derive homology model later on) -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB Figure 3 - BLASTP pairwise alignment between protein tyrosine phosphate in Listeria monocytogenes and 1YWF
---- Query Coverage: 84% ---- Max % Identities: 26% ---- % Positives: 47% ---- Chain used for homology: A
Current Inhibitors: N/A Expression Information (has it been expressed in bacterial cells):http://www.biomedcentral.com/1471-2164/11/457 Purification Method: Glutathione sepharose affinity chromatography Image of protein (PyMol with features delineated and shown separately): N/A *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MKNWVKVTGAGVLSATLLLGGCGAQSEEKAEANVKTEQTLKPGSQIKLEGAVNVRDLGGYKTTDGLTIKPHKLIRSAELANLSDSDKKKLVNTYDLSHIVDFRT SSEVATKPDPKLTDVDYTHDSVMKDNGTSTSTQDLTASLAKMDNPETFLINANKSFITDETSIQAYKDFFDILLANQDGSVLWHCTAGKDRAGFGTALVLSALGV DKNTVIDDYMLSNKYRADENKKAIEAVAAKTDNKKVIDGMTAVMEVRESYINAAFDEINAKYGSMDNFLKEKLGLTDAKKEQLKKAYLY *length of your protein in Amino Acids: 298 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 32585.8 Molar Extinction coefficient of your protein at 280 nm wavelength: 26025 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure 4 - TMpred graph for lmptp
*CDS Gene Sequence (paste as text only):
ATGAAAAATTGGGTAAAAGTAACAGGAGCAGGGGTACTAAGCGCGACTTTACTATTAGGTGGATGCGGGG CGCAGTCAGAAGAAAAAGCAGAAGCAAATGTTAAAACCGAGCAAACACTCAAACCAGGAAGCCAAATTAA ATTAGAAGGCGCTGTAAATGTCCGGGACTTAGGCGGATACAAAACAACGGATGGACTAACCATTAAGCCA CATAAACTCATTAGAAGTGCCGAACTCGCTAACTTAAGTGATTCGGATAAAAAGAAACTCGTAAATACAT ATGATTTGTCTCATATAGTTGATTTCCGAACAAGTTCAGAAGTCGCAACGAAACCAGATCCGAAACTCAC AGATGTAGACTATACGCACGATTCTGTGATGAAAGATAATGGAACATCTACAAGCACACAAGATTTAACT GCTAGCCTAGCCAAAATGGATAATCCAGAGACATTTCTCATTAATGCTAATAAGAGCTTTATTACAGATG AAACTTCGATACAAGCCTATAAAGATTTTTTTGATATACTACTAGCAAACCAAGATGGTTCTGTTCTTTG GCACTGTACAGCTGGAAAAGACCGAGCTGGATTTGGAACCGCCCTCGTTCTTTCAGCGTTAGGTGTGGAT AAAAACACGGTCATTGACGATTATATGCTGTCCAATAAATATCGTGCTGACGAAAATAAAAAAGCAATTG AAGCCGTTGCAGCAAAAACAGATAATAAAAAAGTGATTGATGGAATGACAGCCGTAATGGAAGTTCGTGA ATCTTATATCAATGCAGCCTTCGATGAAATTAATGCAAAATATGGTTCGATGGACAACTTTTTAAAAGAA
AAACTCGGACTAACCGATGCTAAAAAAGAACAACTAAAAAAAGCATATCTTTATTAA
*GC% Content for gene: 37.9%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
1 ATGAAAAACTGGGTTAAGGTTACCGGTGCGGGTGTTCTCTCTGCGACCCTGCTCCTGGGC
61 GGCTGCGGTGCGCAGTCTGAAGAGAAGGCTGAAGCGAACGTCAAAACCGAACAGACCCTG
121 AAACCGGGCTCTCAGATCAAACTGGAAGGTGCGGTAAACGTTCGTGATCTGGGTGGCTAC
181 AAGACCACCGACGGCCTGACCATCAAGCCGCACAAACTGATCCGTTCTGCGGAACTCGCG
241 AATCTGAGCGACTCTGACAAGAAGAAACTGGTTAACACCTACGACCTGTCTCACATCGTT
301 GACTTCCGTACCTCTTCTGAAGTTGCGACCAAACCGGACCCGAAACTGACCGACGTAGAC
361 TATACCCACGATAGCGTTATGAAGGACAACGGTACCAGCACGTCTACCCAGGATCTGACC
421 GCCTCTCTGGCTAAAATGGATAACCCGGAAACCTTCCTGATCAACGCAAACAAGTCTTTC
481 ATTACTGACGAAACGTCTATCCAGGCGTACAAGGACTTCTTCGACATCCTGCTGGCGAAC
541 CAGGACGGTTCTGTTCTGTGGCACTGCACGGCGGGTAAAGATCGTGCGGGCTTCGGCACC
601 GCGCTGGTCCTGTCTGCTCTCGGTGTAGACAAAAACACCGTTATTGACGACTACATGCTG
661 TCTAACAAATACCGTGCGGACGAAAACAAGAAAGCTATCGAAGCGGTTGCGGCGAAAACG
721 GACAACAAAAAAGTTATCGACGGTATGACGGCGGTAATGGAAGTACGTGAATCTTACATT
781 AACGCGGCGTTCGACGAGATTAATGCGAAATACGGCTCTATGGACAACTTTCTGAAAGAA
841 AAGCTGGGTCTGACGGACGCGAAGAAAGAACAACTGAAGAAGGCGTATCTGTACTAA
*GC% Content for gene (codon optimized): 51.5%
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): DNAWorks Results
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): Monica: Forward: TACTTCCAATCCATGAAGAATTGGGTTAAAGTTAC Reverse:
*Target (protein/gene name): Tyrosine Phosphatase
*NCBI Gene # or RefSeq#: Gene #: 985934
*Protein ID (NP or XP #) or Wolbachia#: NP_465325.1
*Organism (including strain): Listeria monocytogenes
Etiologic Risk Group (see link below): Risk Group 2 (moderate individual risk, low community risk)
*/Disease Information (sort of like the Intro to your Mini Research Write up):
L. monocytogenes is a food-borne bacterium that leads to listeriosis; it is the third most common foodborne bacterial pathogen. It can be found in raw vegetables, meat, raw or unpasteurized milk, and dairy products containing such milk. Those at greatest risk include newborn babies (in which listeriosis often results in sepsis and/or meningitis), pregnant mothers, elderly, and those with weakened immune systems, such as HIV patients. Symptoms include nausea, fever, and diarrhea, with a mortality rate of 20-30%. Its fatality rates can surpass those of Salmonella. As a Gram-positive facultative anaerobe, L. monocytogenes is able to survive in both aerobic and anaerobic environments. It expresses the protein beta hemolysin, and is thus able to destroy the host's red blood cells. L. monocytogenes contains D-galactose residues on its surface that interact with D-galactose receptors of the host's intestinal cells, allowing the bacteria to move past the intestinal walls into the host's body. Another protein, internalin, also interacts with intestinal cell proteins to allow entry and invasion. Once it has invaded, the bacterium moves swiftly through the host cell and multiplies, often passing through the blood-brain barrier.
Link to TDR Targets page (if present): N/A
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): http://www.ncbi.nlm.nih.gov/gene/?term=985934
Essentiality of this protein: http://iai.asm.org/content/79/6/2489.full
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): N/A
Complex of proteins?: N/A
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): http://www.jbc.org/content/279/23/24226.long
*EC#: 3.1.3.48
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.3.48
-- Show screenshot of BRENDA enzyme mechanism schematic
Figure 2 - Brenda Reaction Mechanism for Tyrosine Phosphatase protein (EC # 3.1.3.48) from http://www.brenda-enzymes.org/structure.php?show=reaction&id=11748&type=I&displayType=marvin
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Spectrophotometric Stop Rate Determination
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/larprtntyrosinephos.pdf
-- links to assay reagents (substrates) pages.
Imidazole:http://www.sigmaaldrich.com/catalog/product/aldrich/i202?lang=en®ion=US
Phosphatase Substrate:http://www.sigmaaldrich.com/catalog/product/sigma/s0942?lang=en®ion=US
Sodium Chloride:http://www.sigmaaldrich.com/catalog/product/sial/s9625?lang=en®ion=US
DL-Dithiothreitol:http://www.sigmaaldrich.com/catalog/product/sial/d0632?lang=en®ion=US
Ethylenediaminetetraacetic acid disodium salt dehydrate:http://www.sigmaaldrich.com/catalog/product/sial/ed2ss?lang=en®ion=US
Bovine Serum Albumin:http://www.sigmaaldrich.com/catalog/product/sigma/a8022?lang=en®ion=US
Sodium Hydroxide:http://www.sigmaaldrich.com/catalog/product/sial/s5881?lang=en®ion=US
--- List cost and quantity of substrate reagents, supplier, and catalog #
All reagents are from Sigma-Aldrich:
Imidazole: I202-1G ($11.50) = 1 gramPhosphatase Substrate: S0942 ($35.50) = 50 TabletsSodium Chloride: S9625 ($33.90) = 500 gramsDL-Dithiothreitol: D0632 ($41.70) = 1 gramEthylenediaminetetraacetic acid disodium salt dehydrate: ED2SS ($25.70) = 50 gramsBovine Serum Albumin: A8022 ($66.90) = 10 gramsSodium Hydroxide: S5881 ($51.20) = 500 grams
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: Not available (will derive homology model later on)
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
Figure 3 - BLASTP pairwise alignment between protein tyrosine phosphate in Listeria monocytogenes and 1YWF
---- Query Coverage: 84%
---- Max % Identities: 26%
---- % Positives: 47%
---- Chain used for homology: A
Current Inhibitors: N/A
Expression Information (has it been expressed in bacterial cells): http://www.biomedcentral.com/1471-2164/11/457
Purification Method: Glutathione sepharose affinity chromatography
Image of protein (PyMol with features delineated and shown separately): N/A
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MKNWVKVTGAGVLSATLLLGGCGAQSEEKAEANVKTEQTLKPGSQIKLEGAVNVRDLGGYKTTDGLTIKPHKLIRSAELANLSDSDKKKLVNTYDLSHIVDFRT
SSEVATKPDPKLTDVDYTHDSVMKDNGTSTSTQDLTASLAKMDNPETFLINANKSFITDETSIQAYKDFFDILLANQDGSVLWHCTAGKDRAGFGTALVLSALGV
DKNTVIDDYMLSNKYRADENKKAIEAVAAKTDNKKVIDGMTAVMEVRESYINAAFDEINAKYGSMDNFLKEKLGLTDAKKEQLKKAYLY
*length of your protein in Amino Acids: 298
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 32585.8
Molar Extinction coefficient of your protein at 280 nm wavelength: 26025
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure 4 - TMpred graph for lmptp
*CDS Gene Sequence (paste as text only):
ATGAAAAATTGGGTAAAAGTAACAGGAGCAGGGGTACTAAGCGCGACTTTACTATTAGGTGGATGCGGGG CGCAGTCAGAAGAAAAAGCAGAAGCAAATGTTAAAACCGAGCAAACACTCAAACCAGGAAGCCAAATTAA ATTAGAAGGCGCTGTAAATGTCCGGGACTTAGGCGGATACAAAACAACGGATGGACTAACCATTAAGCCA CATAAACTCATTAGAAGTGCCGAACTCGCTAACTTAAGTGATTCGGATAAAAAGAAACTCGTAAATACAT ATGATTTGTCTCATATAGTTGATTTCCGAACAAGTTCAGAAGTCGCAACGAAACCAGATCCGAAACTCAC AGATGTAGACTATACGCACGATTCTGTGATGAAAGATAATGGAACATCTACAAGCACACAAGATTTAACT GCTAGCCTAGCCAAAATGGATAATCCAGAGACATTTCTCATTAATGCTAATAAGAGCTTTATTACAGATG AAACTTCGATACAAGCCTATAAAGATTTTTTTGATATACTACTAGCAAACCAAGATGGTTCTGTTCTTTG GCACTGTACAGCTGGAAAAGACCGAGCTGGATTTGGAACCGCCCTCGTTCTTTCAGCGTTAGGTGTGGAT AAAAACACGGTCATTGACGATTATATGCTGTCCAATAAATATCGTGCTGACGAAAATAAAAAAGCAATTG AAGCCGTTGCAGCAAAAACAGATAATAAAAAAGTGATTGATGGAATGACAGCCGTAATGGAAGTTCGTGA ATCTTATATCAATGCAGCCTTCGATGAAATTAATGCAAAATATGGTTCGATGGACAACTTTTTAAAAGAA
AAACTCGGACTAACCGATGCTAAAAAAGAACAACTAAAAAAAGCATATCTTTATTAA
*GC% Content for gene: 37.9%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
1 ATGAAAAACTGGGTTAAGGTTACCGGTGCGGGTGTTCTCTCTGCGACCCTGCTCCTGGGC
61 GGCTGCGGTGCGCAGTCTGAAGAGAAGGCTGAAGCGAACGTCAAAACCGAACAGACCCTG
121 AAACCGGGCTCTCAGATCAAACTGGAAGGTGCGGTAAACGTTCGTGATCTGGGTGGCTAC
181 AAGACCACCGACGGCCTGACCATCAAGCCGCACAAACTGATCCGTTCTGCGGAACTCGCG
241 AATCTGAGCGACTCTGACAAGAAGAAACTGGTTAACACCTACGACCTGTCTCACATCGTT
301 GACTTCCGTACCTCTTCTGAAGTTGCGACCAAACCGGACCCGAAACTGACCGACGTAGAC
361 TATACCCACGATAGCGTTATGAAGGACAACGGTACCAGCACGTCTACCCAGGATCTGACC
421 GCCTCTCTGGCTAAAATGGATAACCCGGAAACCTTCCTGATCAACGCAAACAAGTCTTTC
481 ATTACTGACGAAACGTCTATCCAGGCGTACAAGGACTTCTTCGACATCCTGCTGGCGAAC
541 CAGGACGGTTCTGTTCTGTGGCACTGCACGGCGGGTAAAGATCGTGCGGGCTTCGGCACC
601 GCGCTGGTCCTGTCTGCTCTCGGTGTAGACAAAAACACCGTTATTGACGACTACATGCTG
661 TCTAACAAATACCGTGCGGACGAAAACAAGAAAGCTATCGAAGCGGTTGCGGCGAAAACG
721 GACAACAAAAAAGTTATCGACGGTATGACGGCGGTAATGGAAGTACGTGAATCTTACATT
781 AACGCGGCGTTCGACGAGATTAATGCGAAATACGGCTCTATGGACAACTTTCTGAAAGAA
841 AAGCTGGGTCTGACGGACGCGAAGAAAGAACAACTGAAGAAGGCGTATCTGTACTAA
*GC% Content for gene (codon optimized): 51.5%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
DNAWorks Results
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Monica:
Forward:
TACTTCCAATCCATGAAGAATTGGGTTAAAGTTAC
Reverse:
TATCCACCTTTACTGTTAGTACAGGTATGCTTTCT